Parker Stephanie A, Diaz Ines Lopez-Calleja, Anderson Kyle A, Batt Carl A
Graduate Field of Biomedical Engineering, Cornell University, Ithaca, NY 14853, USA.
Protein Expr Purif. 2013 Jun;89(2):136-45. doi: 10.1016/j.pep.2013.02.016. Epub 2013 Mar 13.
A single chain variable fragment (J591 ScFv) that recognizes the extracellular glyco-protein prostate specific membrane antigen (PSMA) was designed, constructed, and expressed in Pichia pastoris. Construction of the J591 ScFv was based on the reported complementarity-determining region (CDR) of the PSMA specific J591 monoclonal antibody (mAb). The nucleotide sequence encoding the J591-derived ScFv was codon-optimized for expression in P. pastoris and a 6× his-tag was added to facilitate affinity purification. A down-scale 2L methanol-induced P. pastoris fermentation yielded 330mg of total protein following a 96h induction. Following Immobolized Metal Affinity Chromatography, functionality of the J591 ScFv was confirmed via Western blot, immunoblot, binding studies, and flow cytometry analysis. The J591 ScFv showed binding affinity and specificity to cell extracts containing PSMA and PSMA-expressing prostate cancer cells. Our results demonstrate that functional J591 ScFv can be produced in P. pastoris for use in diagnostic and targeted therapeutic applications.
设计、构建了一种识别细胞外糖蛋白前列腺特异性膜抗原（PSMA）的单链可变片段（J591 ScFv），并在毕赤酵母中进行表达。J591 ScFv的构建基于已报道的PSMA特异性J591单克隆抗体（mAb）的互补决定区（CDR）。对编码源自J591的ScFv的核苷酸序列进行密码子优化以在毕赤酵母中表达，并添加了6×组氨酸标签以促进亲和纯化。在96小时诱导后，2L规模缩小的甲醇诱导毕赤酵母发酵产生了330mg总蛋白。经过固定化金属亲和层析后，通过蛋白质免疫印迹、免疫印迹、结合研究和流式细胞术分析证实了J591 ScFv的功能。J591 ScFv对含有PSMA的细胞提取物和表达PSMA的前列腺癌细胞显示出结合亲和力和特异性。我们的结果表明，功能性J591 ScFv可在毕赤酵母中产生，用于诊断和靶向治疗应用。
